Seventy two cattle with bacterial pneumonia and twelve healthy cattle were studied in detail for haemato-biochemical examination, radiography and tracheobronchoscopy. Haematobiochemical examination revealed leukocytosis with neutrophilia. Radiographic examination showed increased pulmonary infiltration. Tracheobronchoscopic examination of affected animals revealed inflammation, haemorrhage, mucus to mucopurulent exudates in nasal cavity, trachea, bronchi and bronchioles. Bronchoalveolar lavage (BAL) collected through endoscope was subjected to cytology and cultural examination. Cytology of the affected animals showed increased total cell counts and predominant neutrophils.
Pneumonia is one of the important diseases affecting cattle causing severe economic loss to the dairy industry in India. Cattle are more prone for pneumonia due to small physiological gaseous exchange capacity and resultant basal ventilatory activity, increased compartmentalization and reduced alveolar macrophages activity leading to impaired pulmonary clearance mechanism (Radostits
Cattle that were brought to the large animal medical unit of Teaching Veterinary Clinical Complex (TVCC), Veterinary College and Research Institute, Namakkal with clinical signs of fever, nasal discharge, cough and changes in respiratory characteristics were screened. They were subjected to detailed clinical examination, haematological examination, serum biochemical analysis, radiography and tracheobronchoscopy for confirmation of pneumonia.
Tracheobronchoscopy was done in the standing animals without sedation using Olympus™ [CF type V70L] flexible video endoscope with a diameter of 12.9 mm and an usable length of 1680 mm. The endoscope was inserted into the ventral nasal meatus and moved forward along the nasal septum up to the region of the pharynx and nasolarynx. Upon reaching the larynx, the endoscope was inserted into the trachea and moved forward to tracheal bifurcation and to bronchial areas. The following parameters were evaluated in each region: mucosal surface, colour (pink, reddened, anaemic), vascularization, oedema, quantity and a description of any secretions (serous, mucous, mucoid, purulent, mucopurulent or blood mixed) as described by Stierschiender
Bronchoalveolar lavage (BAL) was collected by advancing endoscope as caudal as possible through the bronchiole branches and wedged at the segmental bronchi. About 180 milliliters of normal saline / phosphate buffer saline (PBS) was infused through the working channel of the endoscope and the same was collected in to mucus extractor using a suction apparatus (Kahl and Hofmann, 1985). BAL fluid was divided into two aliquots and subjected to cytological and cultural examinations.
BAL fluid collected for cytological examination was loaded in the WBC counting chamber of haemocytometer under 40x objectives to get the total count of cells (Wilkie and Markham, 1981). Portion of BAL was centrifuged at 900 x g (2000 rpm) for 10 minutes. The supernatant was decanted and the pellet was resuspended in 0.5 ml of saline and was used to prepare smears. These smears were air dried, fixed in the alcohol and stained with Giemsa stain. Slides were evaluated for cytological and differential count evaluation as per Allen
BAL fluid collected aseptically for cultural examination was streaked on blood agar, nutrient agar, brain heart infusion (BHI) agar, cetrimide agar, chocolate agar and MacConkey agar for isolation of bacteria. Part of the BAL was also inoculated on Frey’s mycoplasma medium and then into Frey’s mycoplasma agar for identification of Mycoplasma if any. Culture plates were incubated as per standard methods. Presumptive and definitive identification of pathogens were done by staining characteristics, colony morphology and standard biochemical tests (Barrow and Feltham 1993 and Quinn
Polymerase chain reaction (PCR) was performed using Mastermix (2x Taq MasterMix Red dye - Amplicon, USA) with composition of 150mM Tris Hcl (pH 8.5), 40mM (NH4)2 SO4, 4.0 mM MgCl2, 0.2 % Tween 20, 0.4 mM dNTPs, 0.05 units/µl Taq DNA polymerase, inert red dye and stabilizer and DNA ladder (Bio-basic, USA (100 bp to 1000 bp) and GeNei, Bangalore (50 bp to 500 bp). Individual colonies for different organisms were resuspended in the nutrient or BHI broth and incubated over night. The broth cultures were utilized for the bacterial DNA extraction using the DNA isolation kit (GeNei, Bangalore) and manual heat and thaw method (Radu
Out of 203 cattle screened for various respiratory disorders, 72 cattle were affected with bacterial pneumonia. Twelve apparently healthy cattle were used as control. The predominant clinical signs noticed in cattle with bacterial pneumonia included respiratory distress (97.2 %), increased lung sounds (94.4 %), anorexia (94.4 %), pyrexia (91.7 %), nasal discharge (88.9 %), dyspnoea (86.1 %), muzzle dryness (86.1 %), tachycardia (84.7 %), congested mucous membrane (83.3 %), tachypnoea (79.2 %) and cough (77.8 %). Radostits
Radiography of the affected cattle showed increased pulmonary infiltrations. Thirunavukkarasu
Tracheobronchoscopic examination in cattle with bacterial pneumonia showed mucus, increased fragility of mucosa, petechial haemorrhage / ecchymotic patches, mucoid plugs, mucopurulent exudates with inflammation and swelling of lymphoid follicles in nasopharynx (
Tracheobronchoscopic examination in cattle with bacterial pneumonia.
Bronchoalveolar lavage fluid was collected at site of lesions especially in segmental and subsegmental bronchi of cattle with pneumonia. The organisms responsible for bacterial pneumonia based on cultural examination of BAL fluid in the present study were
Agarose gel electrophoresis pattern showing amplified PCR product of
Agarose gel electrophoresis pattern showing amplified PCR product of
Agarose gel electrophoresis pattern showing amplified PCR product of
Agarose gel electrophoresis pattern showing amplified PCR product of
BAL cytology of cattle with pneumonia revealed increased mean total nucleated cells, macrophages, neutrophils, eosinophils, epithelial cells and plasma cells and decreased mean lymphocytes when compared to respective means of apparently healthy cattle. Thirunavukkarasu
BAL cytology in cattle with bacterial pneumonia. A, Cluster of neutrophils with macrophages (Giemsa stain x1000); B, Sheet of macrophages including binucleate form with stray neutrophils. (Giemsa stain x1000)
In the present study, tracheobronchoscopy facilitated the direct visualization of lesions in respiratory tract of cattle with bacterial pneumonia and collection of BAL at the site of lesions for cytological and cultural examination.
Tracheobronchoscopy has been one of the best diagnostic aids to visualize the respiratory tract with lesions and also useful in isolation of organisms by collecting BAL. Cultural examination of BAL facilitated the etiological diagnosis of bacterial pneumonia. The total and differential cytological evaluations of BAL are useful in the identification of severity of infection in lung.
The authors are thankful to the Dean, Veterinary College and Research Institute, Namakkal and the Director of Clinics, TANUVAS, Chennai for the infrastructure facilities provided during the study.